Methods for characterizing copy number variation using proximity-litigation sequencing

ABSTRACT

Disclosed here is a method for detecting genome rearrangement in a biological sample, comprising: obtaining a contact matrix plotted from proximity ligation sequencing data of at least one chromosome; identifying an abnormal contact pattern in the contact matrix compared to the contact matrix of a reference genome; comparing the abnormal contact pattern in the contact matrix to one or more known patterns associated with genomic rearrangement to identify a type of genomic rearrangement causing the abnormal contact pattern. Also disclosed is a method for detecting genome rearrangement in a biological sample, comprising: selecting linked chromosomal fragments from proximity ligation sequencing data of at least one chromosome, identifying an abnormal covalent bonding pattern of the linked chromosomal fragments compared to a reference genome; and comparing the abnormal covalent bonding pattern to one or more known patterns associated with genomic rearrangement to identify genomic rearrangement causing the abnormal covalent

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/404,176, filed Oct. 4, 2016, which is hereby incorporated byreference in its entirety.

BACKGROUND

Copy number variations are structural variations in the human genomethat play an important role in the development of various diseases andgenetic disorders. Copy number variation may be caused by differenttypes of chromosomal rearrangement events, such as deletion,duplication, and relocation of DNA fragments within a chromosome. Copynumber variation has been associated with various forms of cancer andneurological disorders. Detection of copy number variants of achromosome of interest or a portion thereof in a biopsy sample of apatient can be a powerful tool to identify genetic diagnostic orprognostic indicators of a disease or disorder. Detection of copy numbervariation is also useful in detecting genetic disorders in non-invasiveprenatal testing. The structure of rearrangements is unique to eachpatient and defining its exact nature is relevant for diagnostics andtreatment decisions.

Nuclear proximity ligation assay was first described in Cullen et al.,Science 261:203-206 (1993). Subsequently, nuclear proximity ligation hasbeen combined with high-throughput sequencing to probe three-dimensionalproximity of different genomic segments that are distant from each otheron the one-dimensional linear space of the chromosome. For example,ChIA-PET probes chromatin interaction by paired-end tag sequencing todetect genome wide chromatin interactions mediated by specific proteinfactors, and Hi-C involves high-throughput chromatin conformationcapture for mapping large-scale structures such as topologicallyassociated domains. See Selvaraj et al., Nature Biotechnology31:1111-1118 (2013) and Rao et al., Cell 159:1665-1680 (2014).

SUMMARY

The present inventors successfully applied proximity ligation sequencingin the detection of copy number variations that are characterized byvarious types of chromosomal rearrangement. Accordingly, a first aspectthe invention described herein relates to a method for detecting genomerearrangement in a biological sample, comprising: selecting linkedchromosomal fragments from proximity ligation sequencing data of atleast one chromosome of the biological sample, wherein the selectedlinked chromosomal fragments substantially originate from covalentbonding of two chromosomal fragments; identifying an abnormal covalentbonding pattern of the linked chromosomal fragments compared to areference genome; and comparing the abnormal covalent bonding pattern toone or more known patterns associated with genomic rearrangement toidentify a type of genomic rearrangement causing the abnormal covalentbonding pattern.

In some embodiments, the genomic rearrangement identified is deletion ofchromosomal fragments. In some embodiments, the deletion of achromosomal fragment is identified by: (i) a loss of covalent bondingbetween two or more continuous chromosomal fragments that are linked toeach other in the reference genome, and (ii) a gain of covalent bondingbetween two chromosomal fragments that are separated by one or morecontinuous chromosomal fragments in the reference genome.

In some embodiments, the genomic rearrangement identified is duplicationof chromosomal fragments. In some embodiments, the duplication of achromosomal fragment is identified by: (i) a loss of covalent bondingbetween a first and second chromosomal fragments that are linked to eachother in the reference genome, (ii) a gain of covalent bonding betweenthe first chromosomal fragment and a third chromosomal fragment, whereinthe first chromosomal fragment is separated from the third chromosomalfragment in the reference genome by at least one chromosomal fragment;(iii) a gain of covalent bonding between the second chromosomal fragmentand a fourth chromosomal fragment, wherein the second chromosomalfragment is separated from the fourth chromosomal fragment in thereference genome by at least one chromosomal fragment; and (iv) anenhancement of covalent bonding between two or more continuouschromosomal fragments, from the third chromosomal fragment to the fourthchromosomal fragment, that are linked to each other in the referencegenome.

In some embodiments, the genomic rearrangement identified is relocationof chromosomal fragments. In some embodiments, the relocation ofchromosomal fragments is identified by: (i) a loss of covalent bondingbetween a first and second chromosomal fragments that are linked to eachother in the reference genome, (ii) a gain of covalent bonding betweenthe first chromosomal fragment and a third chromosomal fragment, whereinthe first chromosomal fragment is separated from the third chromosomalfragment in the reference genome by at least one chromosomal fragment;(iii) a gain of covalent bonding between the second chromosomal fragmentand a fourth chromosomal fragment, wherein the second chromosomalfragment is separated from the fourth chromosomal fragment in thereference genome by at least one chromosomal fragment; (iv) a loss ofcovalent bonding between the third chromosomal fragment and a fifthchromosomal fragments, wherein the third and fifth chromosomal fragmentsare linked to each other in the reference genome; (v) a loss of covalentbonding between the fourth chromosomal fragment and a sixth chromosomalfragments, wherein the fourth and sixth chromosomal fragments are linkedto each other in the reference genome; and (vi) a gain of covalentbonding between the fifth and sixth chromosomal fragments, wherein thefifth chromosomal fragment is separated from the sixth chromosomalfragment in the reference genome by two or more continuous chromosomalfragments, from the third chromosomal fragment to the fourth chromosomalfragment.

A second aspect the invention described herein relates to a method fordetecting genome rearrangement in a biological sample, comprising:obtaining a contact matrix plotted from proximity ligation sequencingdata of at least one chromosome of the biological sample; identifying anabnormal contact pattern in the contact matrix compared to the contactmatrix of a reference genome; and comparing the abnormal contact patternin the contact matrix to one or more known patterns associated withgenomic rearrangement to identify a type of genomic rearrangementcausing the abnormal contact pattern.

In some embodiments, the genomic rearrangement identified is deletion ofchromosomal fragments. In some embodiments, the deletion of achromosomal fragment is identified by: (i) a loss of one or more cisinteractions between two or more continuous chromosomal fragments thatare linked to each other in the reference genome, and (ii) a gain of atrans interaction between two chromosomal fragments that are separatedby the two or more continuous chromosomal fragments in the referencegenome.

In some embodiments, the genomic rearrangement identified is duplicationof chromosomal fragments. In some embodiments, the duplication of achromosomal fragment is identified by: (i) a loss of a cis interactionbetween a first and second chromosomal fragments that are linked to eachother in the reference genome, (ii) a gain of a trans interactionbetween the first chromosomal fragment and a third chromosomal fragment,wherein the first chromosomal fragment is separated from the thirdchromosomal fragment in the reference genome by at least one chromosomalfragment; (iii) a gain of a trans interaction between the secondchromosomal fragment and a fourth chromosomal fragment, wherein thesecond chromosomal fragment is separated from the fourth chromosomalfragment in the reference genome by at least one chromosomal fragment;and (iv) an enhancement of one or more cis interactions between two ormore continuous chromosomal fragments, from the third chromosomalfragment to the fourth chromosomal fragment, that are linked to eachother in the reference genome.

In some embodiments, the genomic rearrangement identified is relocationof chromosomal fragments. In some embodiments, the relocation ofchromosomal fragments is identified by: (i) a loss of a cis interactionbetween a first and second chromosomal fragments that are linked to eachother in the reference genome, (ii) a gain of a trans interactionbetween the first chromosomal fragment and a third chromosomal fragment,wherein the first chromosomal fragment is separated from the thirdchromosomal fragment in the reference genome by at least one chromosomalfragment; (iii) a gain of a trans interaction between the secondchromosomal fragment and a fourth chromosomal fragment, wherein thesecond chromosomal fragment is separated from the fourth chromosomalfragment in the reference genome by at least one chromosomal fragment;(iv) a loss of a cis interaction between the third chromosomal fragmentand a fifth chromosomal fragments, wherein the third and fifthchromosomal fragments are linked to each other in the reference genome;(v) a loss of a cis interaction between the fourth chromosomal fragmentand a sixth chromosomal fragments, wherein the fourth and sixthchromosomal fragments are linked to each other in the reference genome;and (vi) a gain of a trans interaction between the fifth and sixthchromosomal fragments, wherein the fifth chromosomal fragment isseparated from the sixth chromosomal fragment in the reference genome bytwo or more continuous chromosomal fragments, from the third chromosomalfragment to the fourth chromosomal fragment.

A third aspect the invention described herein relates to a method fordetecting genome rearrangement in a biological sample, comprising:subjecting the biological sample to proximity ligation sequencing toobtain proximity ligation sequencing data of at least one chromosome ofthe biological sample; selecting linked chromosomal fragments from theproximity ligation sequencing data, wherein the selected linkedchromosomal fragments substantially originate from covalent bonding oftwo chromosomal fragments; identifying an abnormal covalent bondingpattern of the linked chromosomal fragments compared to a referencegenome; and comparing the abnormal covalent bonding pattern to one ormore known patterns associated with genomic rearrangement to identify atype of genomic rearrangement causing the abnormal covalent bondingpattern.

A fourth aspect the invention described herein relates to a method fordetecting genome rearrangement in a biological sample, comprising:subjecting the biological sample to proximity ligation sequencing toobtain proximity ligation sequencing data of at least one chromosome ofthe biological sample; obtaining a contact matrix plotted from theproximity ligation sequencing data; identifying an abnormal contactpattern in the contact matrix compared to the contact matrix of areference genome; comparing the abnormal contact pattern in the contactmatrix to one or more known patterns associated with genomicrearrangement to identify a type of genomic rearrangement causing theabnormal contact pattern.

A fifth aspect the invention described herein relates to a method fordiagnosing a disease or genetic disorder associated with copy numbervariation, comprising: subjecting a tissue biopsy sample of a patient toproximity ligation sequencing to obtain proximity ligation sequencingdata of at least one chromosome of the tissue biopsy sample of thepatient; selecting linked chromosomal fragments from the proximityligation sequencing data, wherein the selected linked chromosomalfragments substantially originate from covalent bonding of twochromosomal fragments; identifying an abnormal covalent bonding patternof the linked chromosomal fragments compared to a reference genome;comparing the abnormal covalent bonding pattern to one or more knownpatterns associated with copy number variation to identify a type ofcopy number variation causing the abnormal covalent bonding pattern andthe location and/or length of the copy number variation, wherein thecopy number variation identified is correlated to a disease or geneticdisorder.

A sixth aspect the invention described herein relates to a method fordiagnosing a disease or genetic disorder associated with copy numbervariation, comprising: subjecting a tissue biopsy sample of a patient toproximity ligation sequencing to obtain proximity ligation sequencingdata of at least one chromosome of the tissue biopsy sample of thepatient; obtaining a contact matrix plotted from the proximity ligationsequencing data; identifying an abnormal contact pattern in the contactmatrix compared to the contact matrix of a reference genome; andcomparing the abnormal contact pattern in the contact matrix to one ormore known patterns associated with copy number variation to identify atype of copy number variation causing the abnormal contact pattern andthe location and/or length of the copy number variation; wherein thecopy number variation identified is correlated to a disease or geneticdisorder.

An addition aspect of the invention described herein relates to use ofproximity ligation sequencing data to reconstruct one-dimensional genomestructure (linear sequence) of a genome that has experienced chromosomalrearrangements.

These and other features, together with the organization and manner ofoperation thereof, will become apparent from the following detaileddescription when taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: PL-Seq results of a continuous chromosome plotted as contactmatrix. The closer two sequences on the same chromosome are to eachother, the more contacts are detected. Most of the interactions arecis-interactions within the same chromosome. A linear chromosome withonly cis-interactions will plot in a contact matrix in a continuouspattern.

FIG. 2: Deletion of a fragment within a chromosome. A deletion generatesone new trans-interaction at reconnection of 5′ and 3′ donor locationthat reveals location of start/end points of the deletion. Allcis-interactions within deleted fragment are lost, which revealsdeletion length. Chromosome sequences are divided into consecutivelynumbered blocks. Block arrows indicate cis- and trans-interactionsbetween sequences blocks, represented by numbered blocks. Lists showlinked fragments as block numbers for both interacting sequencesseparated by a dash. Shown are lists for linked fragments of a wild typechromosome as reference and for the corresponding rearranged chromosome.Thin arrows point at interactions that appear or disappear due to genomerearrangements of various kinds, and connect to the corresponding linkedfragments.

FIG. 3: Contact matrix corresponding to the deletion of a chromosomalfragment as shown in FIG. 2.

FIG. 4: Amplification and insertion of a fragment within a chromosome.An amplification-insertion generates two new trans-interaction locatedat 5′ donor and 5′ acceptor location and at 3′ donor and 3′ acceptorlocation that reveal length and orientation of the duplication. Allcis-interactions within the duplicated fragment are duplicated andquantification reveals fold amplification of duplication. One singlecis-interaction at insertion location is lost which reveals insertionlocation. Chromosome sequences are divided into consecutively numberedblocks. Block arrows indicate cis- and trans-interactions betweensequences blocks, represented by numbered blocks. Lists show linkedfragments as block numbers for both interacting sequences separated by adash. Shown are lists for linked fragments of a wild type chromosome asreference and for the corresponding rearranged chromosome. Thin arrowspoint at interactions that appear or disappear due to genomerearrangements of various kinds, and connect to the corresponding linkedfragments.

FIG. 5: Contact matrix corresponding to the amplification-insertion of achromosomal fragment as shown in FIG. 4.

FIG. 6: Relocation of a fragment within a chromosome. A relocationgenerates multiple changes in a contact matrix, including two newtrans-interactions at 5′ donor and 5′ acceptor location and at 3′ donorand 3′ acceptor location reveal acceptor location, length andorientation of the insert, as well as one new trans-interaction atreconnection of donor 5′ and 3′ end reveals donor location and length.Chromosome sequences are divided into consecutively numbered blocks.Block arrows indicate cis- and trans-interactions between sequencesblocks, represented by numbered blocks. Lists show linked fragments asblock numbers for both interacting sequences separated by a dash. Shownare lists for linked fragments of a wild type chromosome as referenceand for the corresponding rearranged chromosome. Thin arrows point atinteractions that appear or disappear due to genome rearrangements ofvarious kinds, and connect to the corresponding linked fragments.

FIG. 7: Relocation of a fragment within a chromosome. One singlecis-interaction at insertion location is lost that reveals the acceptorlocation. Two cis-interactions at the deletion borders are lost whichreveals donor location. Chromosome sequences are divided intoconsecutively numbered blocks. Block arrows indicate cis- andtrans-interactions between sequences blocks, represented by numberedblocks. Lists show linked fragments as block numbers for bothinteracting sequences separated by a dash. Shown are lists for linkedfragments of a wild type chromosome as reference and for thecorresponding rearranged chromosome. Thin arrows point at interactionsthat appear or disappear due to genome rearrangements of various kinds,and connect to the corresponding linked fragments.

FIG. 8: Contact matrix corresponding to the relocation of a chromosomalfragment as shown in FIGS. 6 and 7.

DETAILED DESCRIPTION

Reference will now be made in detail to some specific embodiments of theinvention contemplated by the inventors for carrying out the invention.Certain examples of these specific embodiments are illustrated in theaccompanying drawings. While the invention is described in conjunctionwith these specific embodiments, it will be understood that it is notintended to limit the invention to the described embodiments. On thecontrary, it is intended to cover alternatives, modifications, andequivalents as may be included within the spirit and scope of theinvention as defined by the appended claims.

In the following description, numerous specific details are set forth inorder to provide a thorough understanding of the present invention.Particular example embodiments of the present invention may beimplemented without some or all of these specific details.

Various techniques and mechanisms of the present invention willsometimes be described in singular form for clarity. However, it shouldbe noted that some embodiments include multiple iterations of atechnique or multiple instantiations of a mechanism unless notedotherwise.

Methods for Identifying Genome Rearrangement and Copy Number Variation

The invention described herein encompasses a method for detecting genomerearrangement and copy number variation in a biological sample usingproximity ligation sequencing data.

In some embodiments, the method comprises subjecting a biological sampleto proximity ligation sequencing to obtain proximity ligation sequencingdata of at least one chromosome of the biological sample. The proximityligation sequencing data can be obtained from, for example, a biologicalsample of a mammal. The proximity ligation sequencing data can beobtained from, for example, a biological sample of a human subject. Theproximity ligation sequencing data can be obtained from, for example, atissue biopsy sample of a human subject.

Specific biological samples include tissue biopsies such as tumorstissues and placenta tissues (NIPT), captured single cells such asnucleated fetal red blood cells in pregnant women's blood (NIPT),circulating tumor cells and circulating immune cells (e.g., to screenfor VDJ recombination in T- and B-cells), as well as cell culturesamples (e.g., to monitor genome integrity of the culture such as stemcell expansion).

Certain embodiments of proximity ligation sequencing are described inSelvaraj et al., Nature Biotechnology 31:1111-1118 (2013) and Rao etal., Cell 159:1665-1680 (2014), which are incorporated by reference intheir entireties. For example, the proximity ligation sequencing cancomprise crosslinking genomic DNAs in situ. The crosslinked DNA can bedigested with a restriction enzyme and ligated to form linked fragments.The linked fragments can be isolated from cells and sequenced to obtainproximity ligation sequencing data. The proximity ligation sequencingdata of a chromosome can be plotted into a contact matrix showing bothlocations of contacts and contact frequencies thereof.

FIG. 1 shows a continuous chromosome plotted as a contact matrix, withchromosomal fragments divided into consecutively numbered blocks, andcontacts between chromosomal fragments highlighted in the contact matrixas pixels. Typically, the closer two fragments on the same chromosomeare to each other, the more contacts are detected. Most of theinteractions are cis-interactions within the same chromosome. A linearchromosome with only cis-interactions will plot in a contact matrix in acontinuous pattern.

Known contact matrix plotted from proximity ligation sequencing data,however, include many pixels that originate not from covalent bondingbetween two chromosomal fragments, but from noncovalent interactionsbetween two distant chromosomal fragments. The contact frequencies ofthese pixels/blocks are typically lower than the contact frequencies ofthose correlating to covalently bonded chromosomal fragments.Accordingly, in some embodiments, the pixels and sequencing readscorrelating to noncovalent interactions between two distant chromosomalfragments are filtered and separated from the pixels and sequencingreads correlating to covalently bonded chromosomal fragments, prior toanalyzing the pixels and sequencing reads for detection of genomerearrangement and copy number variation.

The genome rearrangement and copy number variation can be detected bydirectly analyzing the sequencing reads of proximity ligationsequencing. The method can comprise the steps of: selecting linkedchromosomal fragments from proximity ligation sequencing data of atleast one chromosome of the biological sample, wherein the selectedlinked chromosomal fragments substantially originate from covalentbonding of two chromosomal fragments; identifying an abnormal covalentbonding pattern of the linked chromosomal fragments compared to areference genome; and comparing the abnormal covalent bonding pattern toone or more known patterns associated with genomic rearrangement toidentify a genomic rearrangement causing the abnormal covalent bondingpattern. A reference genome can refer to a genome before the occurrenceof one or more rounds of rearrangement, whereas a sample tested maycomprise one or more rounds of rearrangement.

In some embodiments, a linked-fragments threshold count can be defined,wherein linked-fragments with counts below the threshold are consideredas transient interactions resulting from protein-DNA interactions thatare disregarded, and linked-fragments with counts above the thresholdare considered as permanent DNA-DNA bonds. Next, the linked fragmentsthat originate from newly formed DNA-DNA covalent bonds (rearrangement)can be listed, and rearrangement patterns causative to such linkedfragments can be identified.

The genome rearrangement and copy number variation can also be detectedby analyzing the contact matrix plotted from proximity ligationsequencing data. The method can comprise the steps of: obtaining acontact matrix plotted from proximity ligation sequencing data of atleast one chromosome of the biological sample; identifying an abnormalcontact pattern in the contact matrix compared to the contact matrix ofa reference genome; and comparing the abnormal contact pattern in thecontact matrix to one or more known patterns associated with genomicrearrangement to identify a genomic rearrangement causing the abnormalcontact pattern.

In some embodiments, abnormal contact matrix patterns are recorded. Therecorded patterns are compared to known patterns of specificrearrangements. Recognition of a specific pattern identifies the type ofthe rearrangement(s). The exact location of each recognized abnormalpattern allows identification of exact locations of the observedrearrangement.

The identification of the type, location, length, and/or orientation ofthe genome arrangement are described in detail in the followingparagraphs.

Deletion of Chromosomal Fragments Within a Genome

In some embodiments, the method described herein can be used to detectdeletion of chromosomal fragments.

As shown in FIGS. 2 and 3, a deletion of a fragment within a chromosomecan generate a new trans-interaction at reconnection of 5′ and 3′ donorlocation that reveals location of start/end points of the deletion. Allcis-interactions within the deleted fragment are lost, which revealsdeletion length.

Accordingly, in some embodiments wherein sequencing reads of proximityligation sequencing are directly used to analyze genome rearrangementand copy number variation, the deletion of a chromosomal fragment can beidentified by one or more of: (i) a loss of covalent bonding between twoor more continuous chromosomal fragments that are linked to each otherin the reference genome (e.g., loss of contact between 5 and 6, 6 and 7,and 7 and 8 in FIG. 2), and (ii) a gain of covalent bonding between twochromosomal fragments that are separated by one or more continuouschromosomal fragments in the reference genome (e.g., gain of contactbetween 5 and 8 in FIG. 2).

In other embodiments wherein a contact matrix is plotted from proximityligation sequencing data and used to analyze genome rearrangement andcopy number variation, the deletion of a chromosomal fragment can beidentified by one or more of: (i) a loss of one or more cis interactionsbetween two or more continuous chromosomal fragments that are linked toeach other in the reference genome (e.g., loss of contact between 5 and6, 6 and 7, and 7 and 8 in FIG. 3), and (ii) a gain of a transinteraction between two chromosomal fragments that are separated by thetwo or more continuous chromosomal fragments in the reference genome(e.g., gain of contact between 5 and 8 in FIG. 3).

Duplication of Chromosomal Fragments Within a Genome

In some embodiments, the method described herein can be used to detectduplication (i.e., amplification and insertion) of chromosomalfragments.

As shown in FIGS. 4 and 5, an amplification and insertion of a fragmentwithin a chromosome can generate two new trans-interaction located at 5′donor and 5′ acceptor location and at 3′ donor and 3′ acceptor locationthat reveal the length and orientation of the duplication. Allcis-interactions within the duplicated fragment are duplicated, andquantification thereof reveals fold amplification of duplication. Onesingle cis-interaction at insertion location is lost which revealsinsertion location.

Accordingly, in some embodiments wherein sequencing reads of proximityligation sequencing are directly used to analyze genome rearrangementand copy number variation, the duplication of a chromosomal fragment canbe identified by one or more of: (i) a loss of covalent bonding betweena first and second chromosomal fragments that are linked to each otherin the reference genome (e.g., loss of contact between 4 and 5 in FIG.4), (ii) a gain of covalent bonding between the first chromosomalfragment and a third chromosomal fragment, wherein the first chromosomalfragment is separated from the third chromosomal fragment in thereference genome by at least one chromosomal fragment (e.g., gain ofcontact between 4 and 10 in FIG. 4), (iii) a gain of covalent bondingbetween the second chromosomal fragment and a fourth chromosomalfragment, wherein the second chromosomal fragment is separated from thefourth chromosomal fragment in the reference genome by at least onechromosomal fragment (e.g., gain of contact between 5 and 12 in FIG. 4),and (iv) an enhancement of covalent bonding between two or morecontinuous chromosomal fragments, from the third chromosomal fragment tothe fourth chromosomal fragment, that are linked to each other in thereference genome (e.g., enhanced contact between 10 and 11 and 11 and 12in FIG. 4).

In some embodiments, the duplication of a chromosomal fragment can beidentified by one or more of: (i) a loss of covalent bonding between afirst and second chromosomal fragments that are linked to each other inthe reference genome, (ii) a gain of covalent bonding between the firstchromosomal fragment and a third chromosomal fragment, wherein the firstchromosomal fragment is separated from the third chromosomal fragment inthe reference genome by at least one chromosomal fragment, (iii) a gainof covalent bonding between the second chromosomal fragment and thethird chromosomal fragment, wherein the second chromosomal fragment isseparated from the third chromosomal fragment in the reference genome byat least one chromosomal fragment.

In other embodiments wherein a contact matrix is plotted from proximityligation sequencing data and used to analyze genome rearrangement andcopy number variation, the duplication of a chromosomal fragment can beidentified by one or more of: (i) a loss of a cis interaction between afirst and second chromosomal fragments that are linked to each other inthe reference genome (e.g., loss of contact between 4 and 5 in FIG. 5),(ii) a gain of a trans interaction between the first chromosomalfragment and a third chromosomal fragment, wherein the first chromosomalfragment is separated from the third chromosomal fragment in thereference genome by at least one chromosomal fragment (e.g., gain ofcontact between 4 and 10 in FIG. 5), (iii) a gain of a trans interactionbetween the second chromosomal fragment and a fourth chromosomalfragment, wherein the second chromosomal fragment is separated from thefourth chromosomal fragment in the reference genome by at least onechromosomal fragment (e.g., gain of contact between 5 and 12 in FIG. 5),and (iv) an enhancement of one or more cis interactions between two ormore continuous chromosomal fragments, from the third chromosomalfragment to the fourth chromosomal fragment, that are linked to eachother in the reference genome (e.g., enhanced contact between 10 and 11and 11 and 12 in FIG. 5).

In other embodiments, the duplication of a chromosomal fragment can beidentified by one or more of: (i) a loss of a cis interaction between afirst and second chromosomal fragments that are linked to each other inthe reference genome, (ii) a gain of a trans interaction between thefirst chromosomal fragment and a third chromosomal fragment, wherein thefirst chromosomal fragment is separated from the third chromosomalfragment in the reference genome by at least one chromosomal fragment,(iii) a gain of a trans interaction between the second chromosomalfragment and the third chromosomal fragment, wherein the secondchromosomal fragment is separated from the third chromosomal fragment inthe reference genome by at least one chromosomal fragment.

Relocation of Chromosomal Fragments Within a Genome

In some embodiments, the method described herein can be used to detectrelocation of chromosomal fragments.

As shown in FIG. 6, a relocation of a fragment within a chromosome cangenerate multiple changes in a contact matrix, including two newtrans-interactions at 5′ donor and 5′ acceptor location and at 3′ donorand 3′ acceptor location which reveal acceptor location, length andorientation of the insert, as well as one new trans-interaction atreconnection of donor 5′ and 3′ end which reveals donor location andlength. In addition, as shown in FIG. 7, one single cis-interaction atinsertion location is lost which reveals the acceptor location. Twocis-interactions at the deletion borders are lost which reveals donorlocation.

Accordingly, in some embodiments wherein sequencing reads of proximityligation sequencing are directly used to analyze genome rearrangementand copy number variation, the relocation of a chromosomal fragment canbe identified by one or more of: (i) a loss of covalent bonding betweena first and second chromosomal fragments that are linked to each otherin the reference genome (e.g., loss of contact between 4 and 5 in FIG.7), (ii) a gain of covalent bonding between the first chromosomalfragment and a third chromosomal fragment, wherein the first chromosomalfragment is separated from the third chromosomal fragment in thereference genome by at least one chromosomal fragment (e.g., gain ofcontact between 4 and 10 in FIG. 6), (iii) a gain of covalent bondingbetween the second chromosomal fragment and a fourth chromosomalfragment, wherein the second chromosomal fragment is separated from thefourth chromosomal fragment in the reference genome by at least onechromosomal fragment (e.g., gain of contact between 5 and 12 in FIG. 6),(iv) a loss of covalent bonding between the third chromosomal fragmentand a fifth chromosomal fragments, wherein the third and fifthchromosomal fragments are linked to each other in the reference genome(e.g., loss of contact between 10 and 9 in FIG. 7), (v) a loss ofcovalent bonding between the fourth chromosomal fragment and a sixthchromosomal fragments, wherein the fourth and sixth chromosomalfragments are linked to each other in the reference genome (e.g., lossof contact between 12 and 13 in FIG. 7), and (vi) a gain of covalentbonding between the fifth and sixth chromosomal fragments, wherein thefifth chromosomal fragment is separated from the sixth chromosomalfragment in the reference genome by two or more continuous chromosomalfragments, from the third chromosomal fragment to the fourth chromosomalfragment (e.g., gain of contact between 9 and 13 in FIG. 6).

In some embodiments, the relocation of a chromosomal fragment can beidentified by one or more of: (i) a loss of covalent bonding between afirst and second chromosomal fragments that are linked to each other inthe reference genome, (ii) a gain of covalent bonding between the firstchromosomal fragment and a third chromosomal fragment, wherein the firstchromosomal fragment is separated from the third chromosomal fragment inthe reference genome by at least one chromosomal fragment, (iii) a gainof covalent bonding between the second chromosomal fragment and thethird chromosomal fragment, wherein the second chromosomal fragment isseparated from the third chromosomal fragment in the reference genome byat least one chromosomal fragment, (iv) a loss of covalent bondingbetween the third chromosomal fragment and a fifth chromosomalfragments, wherein the third and fifth chromosomal fragments are linkedto each other in the reference genome, (v) a loss of covalent bondingbetween the third chromosomal fragment and a sixth chromosomalfragments, wherein the third and sixth chromosomal fragments are linkedto each other in the reference genome, and (vi) a gain of covalentbonding between the fifth and sixth chromosomal fragments, wherein thefifth chromosomal fragment is separated from the sixth chromosomalfragment in the reference genome by the third chromosomal fragment.

In other embodiments wherein a contact matrix is plotted from proximityligation sequencing data and used to analyze genome rearrangement andcopy number variation, the relocation of a chromosomal fragment can beidentified by one or more of: (i) a loss of a cis interaction between afirst and second chromosomal fragments that are linked to each other inthe reference genome (e.g., loss of contact between 4 and 5 in FIG. 8),(ii) a gain of a trans interaction between the first chromosomalfragment and a third chromosomal fragment, wherein the first chromosomalfragment is separated from the third chromosomal fragment in thereference genome by at least one chromosomal fragment (e.g., gain ofcontact between 4 and 10 in FIG. 8), (iii) a gain of a trans interactionbetween the second chromosomal fragment and a fourth chromosomalfragment, wherein the second chromosomal fragment is separated from thefourth chromosomal fragment in the reference genome by at least onechromosomal fragment (e.g., gain of contact between 5 and 12 in FIG. 8),(iv) a loss of a cis interaction between the third chromosomal fragmentand a fifth chromosomal fragments, wherein the third and fifthchromosomal fragments are linked to each other in the reference genome(e.g., loss of contact between 10 and 9 in FIG. 8), (v) a loss of a cisinteraction between the fourth chromosomal fragment and a sixthchromosomal fragments, wherein the fourth and sixth chromosomalfragments are linked to each other in the reference genome (e.g., lossof contact between 12 and 13 in FIG. 8), and (vi) a gain of a transinteraction between the fifth and sixth chromosomal fragments, whereinthe fifth chromosomal fragment is separated from the sixth chromosomalfragment in the reference genome by two or more continuous chromosomalfragments, from the third chromosomal fragment to the fourth chromosomalfragment (e.g., gain of contact between 9 and 13 in FIG. 8).

In other embodiments, the relocation of a chromosomal fragment can beidentified by one or more of: (i) a loss of a cis interaction between afirst and second chromosomal fragments that are linked to each other inthe reference genome, (ii) a gain of a trans interaction between thefirst chromosomal fragment and a third chromosomal fragment, wherein thefirst chromosomal fragment is separated from the third chromosomalfragment in the reference genome by at least one chromosomal fragment,(iii) a gain of a trans interaction between the second chromosomalfragment and the third chromosomal fragment, wherein the secondchromosomal fragment is separated from the third chromosomal fragment inthe reference genome by at least one chromosomal fragment, (iv) a lossof a cis interaction between the third chromosomal fragment and a fifthchromosomal fragments, wherein the third and fifth chromosomal fragmentsare linked to each other in the reference genome, (v) a loss of a cisinteraction between the third chromosomal fragment and a sixthchromosomal fragments, wherein the third and sixth chromosomal fragmentsare linked to each other in the reference genome, and (vi) a gain of atrans interaction between the fifth and sixth chromosomal fragments,wherein the fifth chromosomal fragment is separated from the sixthchromosomal fragment in the reference genome by the third chromosomalfragment.

Applications

For diseases and genetic disorders known to be associated with certaincopy number variation such as deletion, duplication, or relocation of achromosomal fragment, the method described herein can also be used fordiagnosing the diseases and genetic disorders.

In some embodiment, the invention provides for a method for diagnosing adisease or genetic disorder associated with copy number variation,comprising: subjecting a tissue biopsy sample of a patient to proximityligation sequencing to obtain proximity ligation sequencing data of atleast one chromosome of the tissue biopsy sample; selecting linkedchromosomal fragments from the proximity ligation sequencing data,wherein the selected linked chromosomal fragments substantiallyoriginate from covalent bonding of two chromosomal fragments;identifying an abnormal covalent bonding pattern of the linkedchromosomal fragments compared to a reference genome; comparing theabnormal covalent bonding pattern to one or more known patternsassociated with copy number variation to identify a type of copy numbervariation causing the abnormal covalent bonding pattern and the locationand/or length of the copy number variation, wherein the copy numbervariation identified is correlated to a disease or genetic disorder.

In some embodiment, the invention provides for a method for diagnosing adisease or genetic disorder associated with copy number variation,comprising: subjecting a tissue biopsy sample of patient to proximityligation sequencing to obtain proximity ligation sequencing data of atleast one chromosome of the tissue biopsy sample; obtaining a contactmatrix plotted from the proximity ligation sequencing data; identifyingan abnormal contact pattern in the contact matrix compared to thecontact matrix of a reference genome; and comparing the abnormal contactpattern in the contact matrix to one or more known patterns associatedwith copy number variation to identify a type of copy number variationcausing the abnormal contact pattern and the location and/or length ofthe copy number variation, wherein the copy number variation identifiedis correlated to a disease or genetic disorder.

In addition, the method described herein can be used for identifying oneor more copy number variations associated with a certain disease orgenetic disorder.

In some embodiment, the invention provides for a method for identifyingone or more copy number variations causing a disease or geneticdisorder, comprising: subjecting a plurality of tissue biopsy samplesfrom patient suffering from a certain disease or genetic disorder toproximity ligation sequencing to obtain proximity ligation sequencingdata of at least one chromosome of the tissue biopsy sample; selectinglinked chromosomal fragments from the proximity ligation sequencingdata, wherein the selected linked chromosomal fragments substantiallyoriginate from covalent bonding of two chromosomal fragments;identifying an abnormal covalent bonding pattern of the linkedchromosomal fragments compared to a reference genome; comparing theabnormal covalent bonding pattern to one or more known patternsassociated with copy number variation to identify a type of copy numbervariation causing the abnormal covalent bonding pattern and the locationand/or length of the copy number variation; and identifying at least onecopy number variation common to a statistically significant number ofpatients suffering from the same disease or genetic disorder.

In some embodiment, the invention provides for a method for identifyingone or more copy number variations causing a disease or geneticdisorder, comprising: subjecting a plurality of tissue biopsy samplesfrom patient suffering from a certain disease or genetic disorder toproximity ligation sequencing to obtain proximity ligation sequencingdata of at least one chromosome of the tissue biopsy sample; obtaining acontact matrix plotted from proximity ligation sequencing data;identifying an abnormal contact pattern in the contact matrix comparedto the contact matrix of a reference genome; and comparing the abnormalcontact pattern in the contact matrix to one or more known patternsassociated with copy number variation to identify a type of copy numbervariation causing the abnormal contact pattern and the location and/orlength of the copy number variation; and identifying at least one copynumber variation common to a statistically significant number ofpatients suffering from the same disease or genetic disorder.

Further Implementations

Many embodiments disclosed herein may be implemented in digitalelectronic circuitry, integrated circuitry, specially designed ASICs(application-specific integrated circuits), computer hardware, firmware,software, or in combinations thereof. Method steps of the presentlydisclosed embodiments can be performed by a programmable processorexecuting a program of instructions to perform functions of thepresently disclosed embodiments by operating on input data andgenerating output; and apparatus relating to the presently disclosedembodiments can be implemented in a computer program product tangiblyembodied in a machine-readable storage device for execution by aprogrammable processor. The presently disclosed embodiments can beimplemented advantageously in one or more computer programs that areexecutable and/or interpretable on a programmable system including atleast one programmable processor, which may be special or generalpurpose, coupled to receive data and instructions from, and to transmitdata and instructions to, a storage system, at least one input device,and at least one output device. Each computer program can be implementedin a high-level procedural or object-oriented programming language or inassembly or machine language if desired; and in any case, the languagecan be a compiled or interpreted language. A computer program may bedeployed in any form, including as a stand-alone program, or as amodule, component, subroutine, or other unit suitable for use in acomputing environment. A computer program may be deployed to be executedor interpreted on one computer or on multiple computers at one site, ordistributed across multiple sites and interconnected by a communicationnetwork.

Computer readable storage media, as used herein, refers to physical ortangible storage (as opposed to signals) and includes without limitationvolatile and non-volatile, removable and non-removable media implementedin any method or technology for the tangible storage of information suchas computer-readable instructions, data structures, program modules orother data. Computer readable storage media includes any type ofnon-transitory computer readable medium including, but not limited to,RAM, ROM, EPROM, EEPROM, flash memory or other solid state memorytechnology, CD-ROM, DVD, or other optical storage, magnetic cassettes,magnetic tape, magnetic disk storage or other magnetic storage devices,or any other physical or material medium which can be used to tangiblystore the desired information or data or instructions and which can beaccessed by a computer or processor.

Any of the methods described herein may include the output of data in aphysical format, such as on a computer screen, or on a paper printout.In explanations of any embodiments elsewhere in this document, it shouldbe understood that the described methods may be combined with the outputof the actionable data in a format that can be acted upon by aphysician. In addition, the described methods may be combined with theactual execution of a clinical decision that results in a clinicaltreatment, or the execution of a clinical decision to make no action.Some of the embodiments described herein may be combined with the outputof the actionable data, and the execution of a clinical decision thatresults in a clinical treatment, or the execution of a clinical decisionto make no action.

In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations, which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scopes ofthis invention.

1. A method for detecting genome rearrangement in a biological sample,comprising: selecting linked chromosomal fragments from proximityligation sequencing data of at least one chromosome of the biologicalsample, wherein the selected linked chromosomal fragments substantiallyoriginate from covalent bonding of two chromosomal fragments;identifying an abnormal covalent bonding pattern of the linkedchromosomal fragments compared to a reference genome; and comparing theabnormal covalent bonding pattern to one or more known patternsassociated with genomic rearrangement to identify a type of genomicrearrangement causing the abnormal covalent bonding pattern.
 2. Themethod of claim 1, wherein the genomic rearrangement identified isdeletion of chromosomal fragments.
 3. The method of claim 2, wherein thedeletion of a chromosomal fragment is identified by: (i) a loss ofcovalent bonding between two or more continuous chromosomal fragmentsthat are linked to each other in the reference genome, and (ii) a gainof covalent bonding between two chromosomal fragments that are separatedby one or more continuous chromosomal fragments in the reference genome.4. The method of claim 1, wherein the genomic rearrangement identifiedis duplication of chromosomal fragments.
 5. The method of claim 4,wherein the duplication of a chromosomal fragment is identified by: (i)a loss of covalent bonding between a first and second chromosomalfragments that are linked to each other in the reference genome, (ii) again of covalent bonding between the first chromosomal fragment and athird chromosomal fragment, wherein the first chromosomal fragment isseparated from the third chromosomal fragment in the reference genome byat least one chromosomal fragment; (iii) a gain of covalent bondingbetween the second chromosomal fragment and a fourth chromosomalfragment, wherein the second chromosomal fragment is separated from thefourth chromosomal fragment in the reference genome by at least onechromosomal fragment; and (iv) an enhancement of covalent bondingbetween two or more continuous chromosomal fragments, from the thirdchromosomal fragment to the fourth chromosomal fragment, that are linkedto each other in the reference genome.
 6. The method of claim 1, whereinthe genomic rearrangement identified is relocation of chromosomalfragments.
 7. The method of claim 6, wherein the relocation ofchromosomal fragments is identified by: (i) a loss of covalent bondingbetween a first and second chromosomal fragments that are linked to eachother in the reference genome, (ii) a gain of covalent bonding betweenthe first chromosomal fragment and a third chromosomal fragment, whereinthe first chromosomal fragment is separated from the third chromosomalfragment in the reference genome by at least one chromosomal fragment;(iii) a gain of covalent bonding between the second chromosomal fragmentand a fourth chromosomal fragment, wherein the second chromosomalfragment is separated from the fourth chromosomal fragment in thereference genome by at least one chromosomal fragment; (iv) a loss ofcovalent bonding between the third chromosomal fragment and a fifthchromosomal fragments, wherein the third and fifth chromosomal fragmentsare linked to each other in the reference genome; (v) a loss of covalentbonding between the fourth chromosomal fragment and a sixth chromosomalfragments, wherein the fourth and sixth chromosomal fragments are linkedto each other in the reference genome; and (vi) a gain of covalentbonding between the fifth and sixth chromosomal fragments, wherein thefifth chromosomal fragment is separated from the sixth chromosomalfragment in the reference genome by two or more continuous chromosomalfragments, from the third chromosomal fragment to the fourth chromosomalfragment.
 8. A method for detecting genome rearrangement in a biologicalsample, comprising: obtaining a contact matrix plotted from proximityligation sequencing data of at least one chromosome of the biologicalsample; identifying an abnormal contact pattern in the contact matrixcompared to the contact matrix of a reference genome; and comparing theabnormal contact pattern in the contact matrix to one or more knownpatterns associated with genomic rearrangement to identify a type ofgenomic rearrangement causing the abnormal contact pattern.
 9. Themethod of claim 8, wherein the genomic rearrangement identified isdeletion of chromosomal fragments.
 10. The method of claim 9, whereinthe deletion of a chromosomal fragment is identified by: (i) a loss ofone or more cis interactions between two or more continuous chromosomalfragments that are linked to each other in the reference genome, and(ii) a gain of a trans interaction between two chromosomal fragmentsthat are separated by the two or more continuous chromosomal fragmentsin the reference genome.
 11. The method of claim 8, wherein the genomicrearrangement identified is duplication of chromosomal fragments. 12.The method of claim 11, wherein the duplication of a chromosomalfragment is identified by: (i) a loss of a cis interaction between afirst and second chromosomal fragments that are linked to each other inthe reference genome, (ii) a gain of a trans interaction between thefirst chromosomal fragment and a third chromosomal fragment, wherein thefirst chromosomal fragment is separated from the third chromosomalfragment in the reference genome by at least one chromosomal fragment;(iii) a gain of a trans interaction between the second chromosomalfragment and a fourth chromosomal fragment, wherein the secondchromosomal fragment is separated from the fourth chromosomal fragmentin the reference genome by at least one chromosomal fragment; and (iv)an enhancement of one or more cis interactions between two or morecontinuous chromosomal fragments, from the third chromosomal fragment tothe fourth chromosomal fragment, that are linked to each other in thereference genome.
 13. The method of claim 8, wherein the genomicrearrangement identified is relocation of chromosomal fragments.
 14. Themethod of claim 13, wherein the relocation of chromosomal fragments isidentified by: (i) a loss of a cis interaction between a first andsecond chromosomal fragments that are linked to each other in thereference genome, (ii) a gain of a trans interaction between the firstchromosomal fragment and a third chromosomal fragment, wherein the firstchromosomal fragment is separated from the third chromosomal fragment inthe reference genome by at least one chromosomal fragment; (iii) a gainof a trans interaction between the second chromosomal fragment and afourth chromosomal fragment, wherein the second chromosomal fragment isseparated from the fourth chromosomal fragment in the reference genomeby at least one chromosomal fragment; (iv) a loss of a cis interactionbetween the third chromosomal fragment and a fifth chromosomalfragments, wherein the third and fifth chromosomal fragments are linkedto each other in the reference genome; (v) a loss of a cis interactionbetween the fourth chromosomal fragment and a sixth chromosomalfragments, wherein the fourth and sixth chromosomal fragments are linkedto each other in the reference genome; and (vi) a gain of a transinteraction between the fifth and sixth chromosomal fragments, whereinthe fifth chromosomal fragment is separated from the sixth chromosomalfragment in the reference genome by two or more continuous chromosomalfragments, from the third chromosomal fragment to the fourth chromosomalfragment. 15-20. (canceled)